Coagulation factors promote brown adipose tissue dysfunction and abnormal systemic metabolism in obesity.

Brown adipose tissue (BAT) has a role in maintaining systemic metabolic health in rodents and humans. Here, we show that metabolic stress induces BAT to produce coagulation factors, which then-together with molecules derived from the circulation-promote BAT dysfunction and systemic glucose intolerance. When mice were fed a high-fat diet (HFD), the levels of tissue factor, coagulation Factor VII (FVII), activated coagulation Factor X (FXa), and protease-activated receptor 1 (PAR1) expression increased significantly in BAT. Genetic or pharmacological suppression of coagulation factor-PAR1 signaling in BAT ameliorated its whitening and improved thermogenic response and systemic glucose intolerance in mice with dietary obesity. Conversely, the activation of coagulation factor-PAR1 signaling in BAT caused mitochondrial dysfunction in brown adipocytes and systemic glucose intolerance in mice fed normal chow. These results indicate that BAT produces endogenous coagulation factors that mediate pleiotropic effects via PAR1 signaling under metabolic stress.

Proteomic analysis of nascent polypeptide chains that potentially induce translational pausing during elongation.

Currently, proteins equipped with "ribosomal arrest peptides" (RAPs) that regulate the expression of downstream genes and their own activity by pausing their own translation during elongation are extensively studied. However, studies focusing on RAP have been conducted primarily in prokaryotic cells; studies on eukaryotic cells, especially mammalian cells, are limited. In the present study, we comprehensively examined translationally arrested nascent polypeptides to gain novel insights into RAPs in mammalian cells. Cetyltrimethylammonium bromide was used to obtain nascent polypeptide chains that were translationally arrested during translation elongation. After proteomic analysis, additional screening by discriminating according to amino acid residues at the C-terminal end revealed several novel RAP candidates. Our method can be applied for comprehensive RAP studies in mammalian cells.

Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines.

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs, DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells, that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and heterogeneous nuclear ribonucleoprotein L [HNRNPL]) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

Hyperactive mTOR induces neuroendocrine differentiation in prostate cancer cell with concurrent up-regulation of IRF1.

BACKGROUND: Neuroendocrine-differentiated prostate cancer (NEPCa) is refractory to androgen deprivation therapy and shows a poor prognosis. The underlying mechanisms responsible for neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the role of mammalian target of rapamycin (mTOR) in NEPCa. METHODS: We utilized a gain-of-function analysis by establishing a human PCa LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR). Then, we employed a comprehensive mass spectrometric analysis to identify a key transcription factor in LNCaP-mTOR, followed by a loss-of-function analysis using CRISPR/Cas system. RESULTS: The activation of mTOR induced NED. We observed significant cell growth arrest in NED of LNCaP-mTOR, which accompanied increased expression of p21WAF1/CIP1 . A comprehensive mass spectrometric analysis identified interferon regulatory factor 1 (IRF1) as a key transcription factor in growth arrest of LNCaP-mTOR. The disruption of IRF1 gene in LNCaP-mTOR reversed cell growth arrest along with the suppression of its target p21WAF1/CIP1 . These results indicate that the growth arrest in NED is at least in part dependent on IRF1 through the induction of p21WAF1/CIP1 . CONCLUSIONS: We identified active mTOR as a novel inducer of NED, and elucidated a mechanism underlying the malignant transformation of NEPCa by recapitulating NED in vitro.

Progerin impairs vascular smooth muscle cell growth via the DNA damage response pathway.

Mutations of the lamin A gene cause various premature aging syndromes, including Hutchinson-Gilford progeria syndrome (HGPS) and atypical Werner syndrome. In HGPS (but not atypical Werner syndrome), extensive loss of vascular smooth muscle cells leads to myocardial infarction with premature death. The underlying mechanisms how single gene mutations can cause various phenotypes are largely unknown. We performed an interactome analysis using mutant forms of lamin A involved in progeroid syndromes. We found that the mutant lamin A responsible for HGPS, known as progerin, could not bind to proteins related to the DNA damage response, including DNA-dependent protein kinase (DNA-PK). In contrast, wild-type lamin A and lamin A mutants causing atypical Werner syndrome were able to bind to these molecules. We also found that forced expression of progerin in vascular smooth muscle cells led to activation of DNA-PK and cellular growth arrest, while knockdown of DNA-PK attenuated this. Deletion of p53 also improved the inhibition of cell growth due to forced expression of progerin. These findings suggested that progerin activates the DNA damage response pathway and that dysregulation of this pathway may be responsible for the development of cardiovascular pathology in patients with HGPS.

Proteomic analysis of immunogenic proteins from salivary glands of Aedes aegypti.

Humans develop anti-salivary proteins after arthropod bites or exposure to insect salivary proteins. This reaction indicates that vector bites have a positive effect on the host immune response, which can be used as epidemiological markers of exposure to the vector. Our previous study identified two immunogenic proteins with molecular weights of 31 kDa and 56 kDa from salivary gland extract (SGE) of Aedes aegypti that cross-reacted with serum samples from Dengue Hemorrhagic Fever (DHF) patients and healthy people in an endemic area (Indonesia). Serum samples from individuals living in non-endemic area (sub-tropical country) and infants did not show the immunogenic reactions. The objective of this research was to identify two immunogenic proteins, i.e., 31 and 56 kDa by using proteomic analysis. In this study, proteomic analysis resulted in identification of 13 proteins and 7 proteins from the 31 kDa- and 56 kDa-immunogenic protein bands, respectively. Among those proteins, the D7 protein (Arthropode Odorant-Binding Protein, AOBP) was the most abundant in 31-kDa band, and apyrase was the major protein of the 56-kDa band.

Human nucleolar protein Nop52 (RRP1/NNP-1) is involved in site 2 cleavage in internal transcribed spacer 1 of pre-rRNAs at early stages of ribosome biogenesis.

During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S.

Human cell growth regulator Ly-1 antibody reactive homologue accelerates processing of preribosomal RNA.

Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly-1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc-finger DNA-binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre-rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre-rRNAs. In addition, we show that over-expression of LYAR increases cell proliferation without affecting the expression of c-Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20°C and 25°C.

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnett's test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.

Splicing factor 2-associated protein p32 participates in ribosome biogenesis by regulating the binding of Nop52 and fibrillarin to preribosome particles.

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.

Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

Automated SPR-LC-MS/MS system for protein interaction analysis.

We have developed a novel automated system to analyze protein complexes by integrating a surface plasmon resonance (SPR) biosensor with highly sensitive nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). A His-tagged protein, which is also tagged with FLAG and biotinylated sequences, was expressed in mammalian cells. After purification by using the His tag from the cell lysate, the sample protein mixture was applied to an SPR biosensor and the protein complex was captured on the sensor chip. The automated SPR-LC-MS/MS was then performed: (1) two-step on-chip purification of the protein complex by using the FLAG and the biotinylated tags, (2) on-chip protease digestion of the complex, and (3) online nanoflow LC-MS/MS analysis of the resulting peptide fragments for protein identification. All of these processes could be monitored in real-time by the SPR biosensor. We validated the performance of the system using either FK506-binding protein 52 kDa (FKBP52) or ribosomal protein S19 (rpS19) as bait. Thus, the fully automated SPR-LC-MS/MS system appeared to be a powerful tool for functional proteomics studies, particularly for snapshot analysis of functional cellular complexes and machines.

Evidence for mitochondrial localization of P5, a member of the protein disulphide isomerase family.

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.

Association of human DNA helicase RecQ5beta with RNA polymerase II and its possible role in transcription.

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.

Hsp90 regulates the Fanconi anemia DNA damage response pathway.

Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG-sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker-induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker-induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47.

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.

Potential roles for ubiquitin and the proteasome during ribosome biogenesis.

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

ERp57 binds competitively to protein disulfide isomerase and calreticulin.

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes.

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.